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«Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory ...»

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Synchronous IPSPs in large groups of BLA principal neurons could also facilitate network oscillations by interacting with intrinsic oscillations in principal neurons to promote rhythmic firing. Synaptic inhibition has previously been shown to shift the phase of intrinsic membrane potential oscillations (MPOs) (Stiefel et al., 2010), suggesting synchronized IPSPs could coordinate oscillations across neurons. Intrinsic MPOs have been shown to improve spiketiming precision (Volgushev et al., 1998; Schaefer et al., 2006), which is, in turn, important for spike-timing dependent plasticity (Dan and Poo, 2004) and signal processing in neural networks (Mainen and Sejnowski, 1995). BLA principal neurons display a highly consistent MPO (Pare and Gaudreau, 1996; Pape et al., 1998) and an intrinsic resonance (Pape and Driesang, 1998), both in the same high delta / low theta frequency band as network oscillations observed during fear learning. If these MPOs were to occur synchronously in groups of BLA neurons, network activity should be promoted at this highly relevant frequency. Considering that groups of cells can have their firing activity entrained by synchronized IPSPs (Hasenstaub et al., 2005), we chose to investigate the possibility that synchronized, rhythmic IPSPs entrain and phase-lock MPOs and coordinate firing activity in BLA principal neurons. Furthermore, we examine the underlying currents and intracellular signaling cascades regulating these phenomena and discuss potential links to synaptic plasticity and fear learning.

6.3 Methods 6.3.1 Animals and housing conditions Whole cell patch clamp recordings were obtained from 76 neurons from 48 rodents, and 46 neurons from 13 primates. Rodent experiments were conducted on tissue from male SpragueDawley rats at 5-7 weeks of age. All rats were group-housed 4 per cage in Plexiglas cages with corn cob (Bed-O-Cob) bedding. Rats had access to food and water ad libitum, and were maintained in a temperature controlled colony room on a 12:12 light:dark cycle. The primate tissue for this study was obtained from juvenile (18-36 months) Macaca mulatta monkeys of both genders. Primates used in this study were born into the breeding colony housed at the Yerkes National Primate Research Center Field Station and raised in normal social groups. They were provided with ad libitum access to food and water and monitored by the Yerkes Veterinary Staff.

Animals used in this study were selected for sacrifice by the veterinary staff for failure to thrive and/or chronic diarrhea refractory to treatment as part of the animal care end-points approved for our monkey colony. Once identified, the animals were moved to the Yerkes Main Station and scheduled for sacrifice within the week.

Experiments for Figures 6.1 & 6.2 were performed in both rat and primate tissue, and the remainder of experiments were performed exclusively in rat tissue (see figure legends for details).

The care of the animals and all anesthesia and sacrifice procedures in this study were performed according to the National Institutes for Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Emory University.

6.3.2 Electrophysiological procedures Preparation of acute BLA slices.

To obtain slices from the rat basolateral amygdala, animals were decapitated under isoflurane anesthesia (Abbott Laboratories, North Chicago, IL). The brains were rapidly removed and placed in ice-cold kynurenic acid-based artificial cerebrospinal fluid (KA-ACSF), which contained (in mM): NaCl (130), KCl (3.5), KH2PO4 (1.1), MgCl2 (6.0), CaCl2 (1.0), NaHCO3 (30), glucose (10), thiourea (0.8), sodium pyruvate (2), ascorbic acid (0.4), and kynurenic acid (2). The glutamatergic antagonist kynurenic acid was included in the KA-ACSF to suppress any excitotoxic effects of glutamate release that may occur due to tissue slicing. A block of tissue containing the BLA was then mounted in a Leica VTS-1000 vibrating microtome (Leica Microsystems, Bannockburn, IL), and 350 µm coronal slices were cut. Slices were hemisected and hand-trimmed to remove excess tissue dorsal to the amygdala. For the primate basolateral amygdala, the animals were sacrificed with an overdose of pentobarbital (100 mg/kg) and handcut blocks of tissue from the medial temporal lobe were mounted in a vibratome and 400 μm coronal slices were cut as previously described (Muly et al., 2009). Slices from both species were transferred to a holding chamber containing KA-ACSF at 32°C and gassed with a 95%/5% O2/CO2 mixture for 40 min before being placed in oxygenated regular ACSF (ACSF) at room temperature containing (in mM): NaCl (130), KCl (3.5), KH2PO4 (1.1), MgCl2 (1.3), CaCl2 (2.5), NaHCO3 (30), glucose (10), thiourea (0.8), sodium pyruvate (2), and ascorbic acid (0.4). Recording procedures.

For recording, slices were placed in a Warner Series 20 recording chamber (Warner Instruments, Hamden, CT) mounted on the fixed stage of a Leica DM-LFS microscope (Leica Microsystems, Bannockburn, IL). Slices were fully submerged and continuously perfused at a rate of 1-2 mL/min with heated (32°C) and oxygenated ACSF. Neurons were selected for recording under IR-DIC illumination with a 40X water immersion objective. Images were captured with a Hamamatsu Orca ER CCD camera (Hamamatsu, Tokyo, Japan) controlled by SimplePCI software (Compix, Sewickley, PA). Whole cell patch-clamp recordings were conducted using thin-walled borosilicate glass-patch electrodes (WPI, Sarasota, FL) which were pulled on a P-97 Flaming / Brown micropipette puller (Sutter Instruments, Novato, CA). Patch electrodes had resistances ranging from 4-7 MΩ when filled with standard patch solution that contained (in mM): K-gluconate (138), KCl (2), MgCl2 (3), phosphocreatine (5), K-ATP (2) NaGTP (0.2), HEPES (10), and biocytin (3 mg/mL). The patch solution was adjusted to a pH of

7.3 with KOH and had a final osmolarity of approximately 280 mOsm. Junction potentials were offset manually prior to patching neurons. Access resistances were monitored throughout recordings and neurons with more than a 15% change were discarded. In the case of paired recordings, two neurons were selected for patching within a single 40X visual field. Neuronal types were pre-selected based on somatic morphology, and type was verified based on electrophysiological profile, as described previously for rat (Rainnie et al., 1993) and primate (Muly et al., 2009).

All recordings were performed in principal neurons of the basolateral nucleus of the amygdala, contained in the basolateral complex. Recordings were obtained using an AxopatchA amplifier (Molecular Devices, Sunnyvale, CA), a Digidata 1320A A/D interface, and pClamp 10 software (Molecular Devices). For all experiments, whole cell patch-clamp configuration was established, and cell responses were recorded in either current clamp or voltage clamp mode. Data were filtered at 5 kHz in current clamp and 2 kHz in voltage clamp, and sampled at a rate of 10 kHz. Neurons were excluded from analysis if their resting membrane potential (Vm) was more positive than -55 mV or if their action potentials did not surpass +5 mV. Drug Application.

Drugs were applied by gravity perfusion at the required concentration in the circulating ACSF. Drugs used: cesium chloride (CsCl), 5 mM; nickel chloride (NiCl2), 500 µM; 4aminopyridine (4-AP), 100-500 µM; tetrodotoxin (TTX), 1 µM; tetraethylammonium chloride (TEA-Cl), 20 mM; forskolin, 10 µM; dideoxy-forskolin, 10 µM; 1,2-bis(o-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid (BAPTA), 5 mM purchased from Sigma–Aldrich (St. Louis, MO); 6,7dinitroquinoxaline-2,3-dione (DNQX), 20 µM; RS-CPP, 10 µM; CGP 52432, 2 µM; 4-(N-ethylN-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7228), 60 µM;

(1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), 50 µM;

8-Br-cAMP, 5-10 µM; and (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium (cAMPs-RP), 25 µM purchased from Tocris (Ellisville, MO). All drugs were stored frozen as concentrated stock solutions in dH2O except DNQX, which was made in 50% dimethyl sulfoxide and buffered to pH 7.3. Spike-timing precision, resonance, and oscillations.

To assess the effect of IPSPs on spike-timing precision, repetitive action potentials were evoked with a depolarizing, square-wave current step of amplitude set to evoke 4-8 Hz firing from a holding potential of -60 mV. The current injections were repeated five times with an interevent interval of 10 seconds. To examine the effect of synaptic inhibition on spike-timing precision, the current in the depolarizing step was transiently removed for 15 ms and then ramped back over 100 ms to the command amplitude to mimic compound spontaneous IPSPs observed in BLA principal neurons. Alternatively, pharmacologically isolated, compound, synaptic IPSPs were evoked using electrical stimulation within the dorsal BLA, just medial to the external capsule. The two IPSPs in each sweep were applied 550 and 1415 ms into the depolarizing step, separated by 865 ms start-to-start (~1.2 Hz), to mimic the frequency of spontaneous IPSPs previously observed in our laboratory.

To examine the membrane potential oscillation of BLA principal neurons, cells were held at -60 mV and injected with the same transient (2.5 s) square-wave depolarizing current pulse as described above. TTX (1 µM) was included in all experiments investigating the membrane oscillation. The voltage response to the DC current pulse was recorded and characterized in regular ACSF and also in varying drug conditions. The amplitude of the current pulse was adjusted such that the steady state membrane potential achieved during current injection was similar before and during drug application (between -40 and -30 mV). Any drug-induced changes in resting membrane potential were compensated for by DC current injection before initiating the transient square-wave depolarizing current pulse to assess the effect on membrane potential oscillations. To assess resonance frequency, principal neurons were held at -60 mV with DC current injection and a sinusoidal frequency sweep of constant current amplitude was injected, increasing from 1-12 Hz over a period of 8 seconds, and the voltage response of the cell was recorded.

6.3.3 Data and statistical analysis The correlation of spontaneous IPSPs and burst-firing from paired recordings of BLA neurons were analyzed by first identifying event times using pClamp software and then using a Pearson product-moment correlation. Spike-timing precision was assessed using a correlationbased metric adapted from Schreiber et al. (2003). The correlation statistic (Rcorr) was calculated for windows of 200 ms every 66 ms, using the equation (Equation 6.1) as published. Briefly, spike times from N traces were convolved with a Gaussian filter of pre-determined width (σ) to create spike vectors (s). For experiments involving artificial and evoked IPSPs, σ = 6 ms, and to prevent a floor effect due to lower spike rates, for experiments with spontaneous IPSPs, σ = 20 ms. The degree of correlation between the vectors (si, sj) was calculated using a dot-product

–  –  –

When calculating spike-timing precision within cells, all 5 traces (N = 5) were compared using this algorithm; when calculating across cells, only the 2 traces (N = 2) which occurred simultaneously were compared, and 5 comparisons were made and averaged. Statistical analyses were performed using a two-way Analysis of Variance (ANOVA), with Bonferroni post-tests to compare across windows and conditions.

Oscillations of the membrane potential of BLA principal neurons were analyzed by means of multi-taper spectral analysis using a custom program that was modified from the Chronux toolbox (Mitra and Bokil, 2008). The resonance frequency of BLA principal neurons was analyzed with fast Fourier transforms (FFT) in pClamp 10 (Molecular Devices) using a Hamming window. Power spectra (mV2/Hz) were converted into standardized Z-scores and peak amplitudes were analyzed using a one-way ANOVA.

6.4 Results 6.4.1 Primate BLA principal neurons receive spontaneous, synchronized, rhythmic IPSPs that coordinate action potential timing.

We have shown previously that approximately 80% of principal neurons in slice preparations of the rat BLA receive spontaneous, compound IPSPs that occur rhythmically at frequencies ranging from 0.5-2 Hz, with a mean of 1.2 Hz, in control ACSF (Rainnie, 1999b).

These compound IPSPs were observed in principal neurons with varying intrinsic properties (mean ± SD: input resistance 85 ± 28MΩ, action potential threshold -43 ± 3.5mV, action potential half width 0.8 ± 0.1ms, data not shown). Here we show that compound IPSPs are also observed in 67% of primate BLA slices with a frequency of 0.76 ± 0.33 Hz, similar to the rat (n = 46, Figure 6.1A, B). As in the rat BLA, compound IPSPs in the primate BLA were highly rhythmic, with a coefficient of variation of instantaneous frequency of 0.30 ± 0.09 (n = 12).

Compound IPSPs occur synchronously across multiple neurons in the rat BLA (Rainnie, 1999a;

Popescu and Pare, 2011), and new analysis reveals they have a near perfect correlation in time across pairs of principal neurons (Pearson product-moment correlation, R2 = 0.999; n = 11, data not shown). We extend this observation to show that compound IPSPs are also highly synchronized across pairs of primate neurons (Figure 6.1A; Pearson product-moment correlation, R2 = 1.0; n = 5, data not shown), suggesting this is an evolutionarily conserved phenomenon.

Using paired recordings from a burst-firing interneuron and a principal neuron, we extend previous observations in the rat BLA (Rainnie, 1999a; Popescu and Pare, 2011) to the primate.

Here we show that compound IPSPs observed in BLA principal neurons (Figure 6.1C, upper trace) coincide with rhythmic bursts of action potentials occurring in burst-firing interneurons (lower trace, n = 2; Pearson product-moment correlation, R2 = 0.999, data not shown), which we have previously shown in the rat BLA to express the calcium-binding protein PV+ (Rainnie et al., 2006). Figure 6.1D illustrates a typical burst-IPSP pair at higher temporal resolution.

Compound IPSPs with a similar waveform can also be observed in principal neurons if an interneuron is driven to fire bursts of action potentials by direct current injection (Figure 6.1E).

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